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the chddren s memorial hospital 2300 children's plaza chicago, illinois 60614 312 ~80-4t~3 February12,1996 The Council for Tobacco Research - USA, Inc. Supporting Biomedical Investigation 900 3rd Avenue New York, NY10022 Tel: (212)421-8885 RE: .~ ":-,,6an for Research Support Dear Sir/Madam: Enclosed please find a copy for Tobacco Research for con Title of the project: Induct Actio Phone number: (312)880. Duration of the project: ' The estimated first year', If you will have any ques in the above address. Thank you. it to The Council .echanisms of eel free to contact me Sincerely, Yuqi Zhao, Ph.D.

,children's memorial hospital 23C~3 children% plaza chicago, illinois @3614 312 8813-4000 TeL. (312)C-<30-~605 Fa~: (3121~0-6609 February 12, 1996 The Council for Tobacco Research - USA, Inc. Supporting Biomedical Investigation 900 3rd Avenue Nexv York, NY10022 Tel: (212)421-8885 Application for Research Supp,-~rt Dear Sir~,iadam: Enclosed please find a copy of the research grant proposal I wish tu submit it to The Council for Tobacco Research for consideration of a two-year research grm:t. Title of the project: Induction of Cell Cycle G2 Arrest by HIV-I Vpr: Mechanisms of Action. Phone number: (312)880-6608; Fax number: (312)880-6609 Duration of the project: Two years The estimated first year's annual direct cost: $75,000 If you will have any questions with regard to this application, pleases feel free to contact me in the above address. I am looking forward to hearing from you soon. Thank you. Sincerely, Yuqi Zhao, P~,.D.

Project Description Title Induction of cell cycle G2 arrest by H~r-1 Vpr: dechanisms c~f :~c~i~n Abstract Recent studies showed that HIV-1 Vpr induces cell cycle G2 arrcs: by mediating host cell cycle control process (Rogel et al., 1995; Jowett et al., 995). Studies t'ur~iacr dernonstmted that Vpr inactivates p34*~'~/cyclin B complex by perturbin. ~ upstream regulators of this complex (Re et al., 1995; He et al., 1995). In this stud3', we will use he fission yeast &'hic,~saccharomycespombe as a model system to investigate the specific time :ion of HIV-1 Vpr in association with cell cycle G2 arrest. Cell cycle G2/M checkpoint control is a highly conserved process among all eukaryotes. In fact, a number of cell cycle control genes are interchangeable between human and fission yeast. Our preliminary study showed that HIV-1 Vpr induces 152 ma'est in b: pombe. Other cellular changes (e.g., morphological chan~ es and growth dela.v~ induced by HIV-1 Vpr in S. pombe also mimicked those observed in humar cells. Theretbre. S. pombe provides a unique model system for us to study HIV-1 Vpr function in mediating host cell cycle control process. Our specific aims are I> to determine which cell ,;ycle control pathxvay Vpr mediates to induce cell cycle G2 arrest, and 2> to identify the cellula" protein(s) that Vpr directly interacts with in the G2-inducing process. Background and Significance Function of the Vpr Vpr affects various human cellular function~, including prevention of cell proliferation (Rogel et al., 1995), changes in cell ~norphology (Levy' ctal., 1993; Re et al., 1995) and induction of cell cycle G2 arrest (Jowett et al., 1995; He et al.. ~,mS. Re et al., 1995). Mechanistically, Vpr induces G2 arrest by inhibit ng the activities of p34':'t~-~ and cyclin B (Re et al., 1995; He et al., 1995), two highly conserved 1: roteins that control cmry of mitosis or G2 arrest in all eukaryotic cells (Norbury and Nurse, t989; Morgan, 1995). These results suggested that effect ofI-~V-1 Vpr on cell cycle G2 arrest is mediated through a common cellular process and should be highly conserved between human and S. pombe (see the Table below). Use of fission yeast as a model system to study the role of Vpr in cell cycle G2 arrest Fission yeast is a monocellular eukaryote, which resembles higher eukatTotic cells in many respects. For example, it divides in a binary fashion and has a .typical cell cycle. A S pombe chromosome replicates autonomo~Iy in mouse cells (Allshire et al., I987). Many mammalian genes can be properly expressed in S pombe cells using • I ~. pombe Mammalian References genes homologues c :lc2 Human CDC2Hs Mouse CDC2 c:lc13 Human cyclin B c~lc25 Human CDC25Hul Human CDC25Hu2 Mouse homolg weel Human tVEE1Hu Mouse s-~c l Htmaar~ rTd9 R~touse r :d24/25Htmaan 14-3-3 Lee & Nurse, 1987 Spun: et al., 1990 Ozon 1991 Sadhu et al., 1990 Nagata et al., 1991 Okazaki et al., 1990 Igarash~i et al., 1991 Okazaki et aL, 1990 /~Jchardson et aL I990 Zhao et al.~ 1992 Ford et aI. 1994

).ea~.t or human viral prorr oters (including HIV LtR and ye~t ~,~ :' pa,moters), or vi:e ve~s~ ITo,.ama et al., 199,; Mat ndreI1. 19Dl.ll. The conser'¢ation of cell cx tic control pro:ess l:et'.'.e<~~. higher e~:aryotes and S ,~ ombe can t:e clearly demons'~rated by functional comptemertation of a number ofS. pombe cell c ¢cle control mntank,~ by human hamologu~'s (see the above "l'able). Furthermore, S'. pornbe is a haploid organism, genetically tractable. :rod amendable to many lnolecular and laboratory manipulations (for reviews see, Zhao and Licb~:rman, 1993, 995). In fact, a wide array ofS. po~rbe mutants, that are defective in pathwa.vs leading to cell Cl.,cle G2 arrest, are available for th~ proposed study (see the Figure below). Tl~erefore, S. pombc provides a unique system for us to determine which cell cycle control path~ay Vpr mediates to trigger cell cycle G2 arrest. In additk.n, the use of a protein-protein interaction detection assay (t~ co-hybrid system) allo~vs us to ident:fy the specific cellular proteins that inter~,:~ directly with Vpr in G2 induction. Cell cycle control patln, ~vs that could lead to G2 arrest Mutation_-_ iu any of the fol owing key cellular components or regulatory pathways will lead to cell cycle G2 arrest in S. poml e cells: ¢dc2 p34 /¢yehn B complex:. Association of p34'~¢-~ to cyclin B and its j~hosphorylation status determines the onse. of mitosis or G2 arrest. p_34eae2 regulators: cdc25: a mitotic inducer wee1 and mikl: mitotic inl,ibitors pp2A: regulator of cdc25 and wee I Control Genes radl, 3, 9,17 husl, rad26 The G2/M checkpoint con rol pathway: ~ At least nine genes have bt:en identified in this pathway. The relationships between these genes are unknown. They may function in m orderly or complex process. .Cyelin B proteo|ysis:, bus.; gene may be involved in degradation of cyclin B, which is required for exit from mitosis (AI-I~ hodairy et al., 1995; Seufert et al., 1995 ~ Broad goals and specific aims The speeitie aims of this ~tudy are 1> to determine which cell cycle control pathway -IIV-1 Vpr mediates and, 2> to identit~r the specific cellular protein(s) Vpr interacts with in the process of ceil cycle G2 arrest induction. A number of recent studies have suggested that Vpr may play an important role in th~ HIV pathogenesis by preventing cell proliferation (Rogel et al., 1995), activating viral replit:ation (Marzio etal., 1995) and r.:gulating viral latency (Le'.2¢ et al., 1994~, Hence, the long-term goaI~ of this project are to under ;land the nature of HIV-I Vpr action in pzrturbing cellular 1: rocess, and to search for ways to l:lock Vpr-induccd cellular catastrophe and viral replication. By using the described S pombe syr tern, we will be able to narrow down which cellular G2-con rolling pathv, ay Vpr mediates and ultimately, to assign its specil~c interact i ~,~ ~s ~.~ ~th ho~ cellular

CD-RP.E~-,rrLY ACTIVE G~TTS, CON-TIL~CYS ~d OTI~R SOLACES of~S Li~ ~ci~ ~ppo~ (~e~ co~, o~y) from aI~ sources, incladi~ o,,~ institution. Sources Totfl V~ue Cu~ent Title of Project Junior Faculty Award numbers) Chicago Pediatric Faculty Foundation Date of of Cu-ant (direct co.-m) $135,000 Annual Amoum Available to You $~5,000 Termination of Grant 7/30/97 Identify. and describe any overlap oftMs application with the above grants: Part of th~s fund ~s used to initiate the proposed pcoject. Indicate the total annual funds available to you this year for all researci~ ! projects under your supervision. I $ 45,000 PENDING OR PLANNED Title of Project Sources Total Value Avg..Annual Total (give grant of Grant Amount Duration numbers) (direct costs) Available to You (give inclusive dates) RadiatEon Mutagenes~s in a New Gene System NIH ROI 034,357 $!I0,000 Identi~ and describe any overlap of this application with the above project, 7/1/96-6/30/2( There is no scientific nor budgetary.-" overlap ~rith tl~c proposed project. 100

proteins inv,~Ived in inductit~n of cell cycle G2 acre.q. -l~e obtainc,i rc.,ults trc, m th~s pr,~ecl v.ilI be m~dotabte_-11y invaluable in heIping us to understand the sFecific iclc c f Vpr in patlaogenesis of HIV-I infee ion. Suppnrting data In a prelin inary study, we successfully cloned and expressed tl~e I-II V-1 Wr gone in ~,2 p~mlb,' cells as conl~rmed by immunoblot analyses. The ~r gone was clox:¢d into tlxree derivatives of a yeast expression vector pREP ~.~asi et al., 1993). Each derivatix c ~ .,pro: sos ~7)t" gone at different levels. By using an inducible nmtl promoter (Maundrell, 1990). ~vc were also able to induce and repress HIV-I ~7~r gene expression in the presence (~pr gone-OFF) and absence (~pr gene-ON) of thiamine in growth media. This feature allows us to test the Vpr-spccifit effects on cellular functions by switching the ~pr gene ON or OFF. Our results indicmed that Vpr indeed induce cell cycle G2 arrest in S. pombe cells as determined by flow eytometry analyses and DAPI nucleus stairdng method. In addition, the effects of Vpr on other cellular changes of S. po,nbe also mimicked those of observed in human cells. Namely, Vpr altm cd cell morphology and prevented cell proliferation. Vpr's effects on these cellular changes in X pombe was lbund to correlate with the levels of vpr gone expression. Our results valida~c~i th: utility ors. ,oombe as a useful model system to investigate HIV-1 ~pr gone functions. Resulzs of this preliminary study have been submitted for publication (Zhao et al., 1995, see the enc!oscd manuscript). Experimental Design and Procedures Other studies by coimmunoprecipitation (Re et al., 1995) and imn',ul~oblot analyses (He et al., 1995) sugge:;ted that Vpr does not interact directly with p34~'~/cycim B :omplex, indicating Vpr may perturb upstream regulators of this complex. We hypothesizc that Vpr either activates mitotic inhibitors (i.e., ~veel and mikl) or inhibits a mitotic induce'. (i.e.. cdc25, pp2A). This could be a direct protein-protein interaction or through one of the several G2-inducing pathways (Hypothesis A). Alternatively, Vpr could also act as m~ independc~ 4 mitotic ilthibitor. It may interact with other gene products that are currently unrecognized (ttypothesis B). To test the first hypothesis, we will express the vpr gene under various cell cycle G2 arrest- deficient backgrounds (Refer to the figure for a list of these mutants avaitable for this study). Suppression of Vpr-induced G2 arrest under a specific gone deficient background will allow us to determine what type of the genetic defects or which cell cycle control pathway Vpr is involved in inducing (}2 arrest. The cell cycle G2 arrest will be measured by flow cytometry and confirmed by a conventional DAPI staining method. Phosphorylation status of the p34~°~ will be determined using an immunoblot analysis method described by Hayles m~d Nurse, (1995). To test the second hypothesis, we will use a yeast two-hybrid system (originally described by Fields and Song, 1989. Test kits are now commercially available f~r ,S' l~ombe from several Bio- teeh companies). This assay is specifically designed for genetic screening of protein-protein interactions. It has been used widely for various eukaryotic systems and proven useful for identifying h~teractions between a known protein and previously unidentified proteins. To prepare for this assay, we have subeloned the HIV-1 vpr gone int~ a bait plasrnid pGBTg, which ~,dll be used in this system as a target for potential cellular protein bindings. 505~08

Biographical Sketch (Yuqi Zhao) Assistant professor, Department of Pediatrics, Net'&western University Medical School Director, Molecular Diagnostics Lab., I-IW Program for Children and Mothers, The Children's Memorial Hospital, Chicago, 1994-present Research .Associate Scientist, Department of Radiation Oncology, College of Physicians & Surgeons, Columbia University, 1992-1994 Education and professional training: Postdoc., College of Physicians & Surgeons, Columbia Univ. (Moi. biology), 1991-92 Ph.D., Oregon St. University (Microbial genetics and molecular biology), 1991 M.S., Oregon St. University (Genetics), 1985 B.S., Shandong College, China (Biology), 1981 Pertinent Editorial and Professional Services: Member of HIV Drug Resistance Sequencing Team, AIDS Clinical Trial Groups (ACTG), 94-present Member of HIV in situ Drug Resistance Screening Team, ACTG. 1995-present Consultant for biological sciences, Science Press, New York Ltd., 1996- Member of editorial board, Clinical Immunology Newsletter, 1995-present Member of editorial board, Journal of Bio-seienee and Technology, 1993-1994 Member of editorial board, Current Ag. Science and Technology. 1990-1994 Membership of professional societies: The AIDS Clinical Trail Group (ACTG) American Society for Microbiology American Association for the Advancement of Science American Pediatric Infectious Diseases Society American Radiation Research Society Relevant and/or Recent Scientific Publications: Zhao, Y., J. Cao, J. Tian, M.ILG. O'Gorman and R. Yogev. 1996. HIV-I ~pr gene induces changes of cell morphology and cell cycle 132 arrest in fission yeast Schizosaccharomycespombe. Presentation at 3rd Conference on Retorviruses and Opportunistic Infections, Washington. D.C. $8-21. Zhao, Y,, J. Cao, J. Tian, M.1LG. O'Gorman and R. Yogev. 1996. Expression of HIV-1 ~77r gene induces changes in fission yeast Schizosaccharom3'ces pombe similar to those induced in human ceils (Abst). Keystone Syrup. on Molecular Biology of Hrv. Taos, NM. B1.-455.

Zhao, Y., J. Cao, NI.R.G. O'Gorrns.n -and R. Yogev. 1995. Effect c, fhurnan immunedeficieney viru~ t)l~e 1 protein R (~:pr) gene exprezsion on basic celluI~ function of fission yeast Schizosaccharomyces pombe tSubmitted). Zhao, Y., W. Kabat, T. Yeghiazarian, J. W'flber and R. Yogev. 1 qq5. Presence ofnon- ,Arion associated p24 antigen of human immunodeficiencv virus type 1 (HI¥-1) in plasma of HIV-irtfeeted eb, ildren (Submitted). Zhao, Y. and H.B. Liebermann. 1995. Schizosaccharomycespombe: a model system for molecular studies ofeukaryotie genes. DNA and Cell Biology 14(5):359-371. Li, Y. and Y. Zhlo (eds.) 1995, 1996. Practical Protocols in Molecular Biology. Science Press, New York Ltd. and Academia Sinic~ Beij ing. Kabat, W., T. Yeghiazafian, J. Wilber, Y. Zhao, and 1L Yogev. 1995. The use era modified branehed DNA signal amplification method to measure human immunodefieieney vires type 1 plasma viral RNA in pediatric population (Abst). Nineteenth AIDS Clinical Trails Group Meeting, XVashington, DC., Abstract #8. Yeghiazarian T, W. Kabat, J. Wilber, Y. Zhao, R. Yogev. 1995. Quantitation of human immunodefieieney virus type 1 (HIV-1) RNA level in pediatric population using a modified branched DNA signal amplification method (Abst). [CAAC, San Francisco, CA, 1-161, p.233. DeMeter L, A Erice, D Mayers, ... Y. Zhao, et al., 1995. Interlaboratory concordance of DNA sequence analysis of the HIV-1 reverse transcdptase (Abst). ICAAC, San Francisco, CA, 1-284, p.256. Zhao, Y. and S. Liu. 1995. The use of oneogenes and tumor suppressor genes as the biomarkers for the early detection of the human colon cancer. Clinical Immunology Newsletter 15(5):65-69. Zhao, Y. and W. Kabat. 1994. New viral markers and their detection methods for clinical studies of human immtmodeficieney virus type !. Clinical Immunology Newsletter. 14(7): 111-117. Zhao, Y., L. Godparthi, and H.B. Liebermann. 1994. A new shuttle vector system for the identification of spontaneous and radiation-induced mutations in the fission yeast Schizosaccharomycespombe. Mutation Res. 311:111-123. Zhlo, Y. and H.B. Liebermann. 1993. The isolation and analysis of mammalian genes using yeast as a host for cloning and expression. J. ofBio. Sci. & Teeh. 1:20-27. Zhao, Y., K.M. Hopkins, and H.B. Liebermann 1993. A method lbr long-term storage and high efficiency transformation of competent fission yeast cells. BioTeehniques. 15(2):238. Zhao, Y., K.M. Hopkins, and H.B. Lieberman. 1992. Identification and isolation of the mammalian cognate of the Schizosaccharomycespombz tad9 gene. CRR Report, Columbia Universit?,.,. 111-114. Romantschuk, M., Y. Zhan, K. McCluskey, J. Williams, and D. Mills. 1990 Repetitive sequences in Pseudomonas syringae pv. phaseolicota: distribution and possible function as insertion sequences. Symbiosis. 595=~10

Effect of Human Immunodeficiency Virus Type 1 Protein R (~p/l Gene Expression on Basic Cellular Function of Fission Yeast Schizosaccharomyces pombe Running title: HIV-1 vpr gene expression in fission yeast Yuqi Zhao*~, Jian Cao, Maurice R.G. O'Gorman, and Ram Yogev HIV Program for Children and Mothers, The Children's Memorial Hospital, Chicago, and Department of Pediatrics, Northwestern U~versity Medical School ~Corresponderxt Footnote: * Corresponding author. Mailing address: 24-30 N. Halsted Street, #218, Chicago, IL 606I~-. Phone: (312)880-6608. Fax: (312)~0-6609. Electrode mail address: yzhao~wu.edu

2 Abstract The HIV-1 vpr gene has been reported effect basic human cellular function, including prevention of cell proliferation, changes in cell morphology and ~nducz[on of cell eyele G2 arrest. : In this study, we used the fission yeast Schizosaccharomycespombe as a model system to investigate the cellular effects of HIV-1 vpr gene expression. The ~r gene was cloned into three derivatives of an inducible gene expression vector pREP. Each derivative allows different level of basal gene expression that can be repressed or induced in the pre~ence or absence Of thiamine. Induction of HI-V-1 vpr gene affects S. pombe at colony, cellular and molecular levels. Specifically, Vpr induces reduced colony size, high diversified ce!l morphology, ~owth delay and cell cycle G2 arrest. These phenotypie changes correlated with the levels of vpr gene expression. Our results suggest that HIV- I Vpr-indueed cellular changes i~ S. pombe rrtmAe those observed in human cells. Therefore, S. pombe system is suitco tbr investigation of the HIV-1 vpr gene function. 505~4412